Systems, methods, compositions and solutions for perfusing an organ

ABSTRACT

The invention, in various embodiments, provides systems, methods and solutions for perfusing an organ.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. patent application Ser. No.11/246,919, filed on Oct. 7, 2005; U.S. Provisional Patent ApplicationSer. No. 60/694,971, filed on Jun. 28, 2005; and U.S. Provisional PatentApplication Ser. No. 60/725,168, filed on Oct. 6, 2005, and entitledSystems and Methods for Ex-Vivo Organ Care. The specifications of eachof the foregoing are incorporated by reference herein in their entirety.

FIELD

The invention relates generally to organ perfusion. In variousillustrated embodiments the invention provides for systems, methods,compositions and solutions for perfusing an organ.

BACKGROUND

Many patients need organ transplants but are unable to obtain suitableorgans. One of the primary reasons is that organs that are otherwiseviable when harvested from a donor are not preserved for long enoughperiods of time to allow them to be transported to appropriaterecipients. With current preservation techniques, transplantable organsremain viable for about three to four hours and, beyond that time,suffer ischemia and tissue injury, which renders them unviable fortransplant.

Current preservation techniques include cryopreservative methods, whichinvolve the cooling of the transplantable organ to temperatures wellbelow physiological temperatures (e.g., below 25° C.). Such techniquestypically use preservation solutions that do not replenish energysources within the organ during transplant or maintain the organ in afunctioning state and therefore are largely ineffective in preventingischemia and other injuries to the organ. The solutions also often relyon the use of high molecular weight impermeants to maintain the organ atthese temperatures prior to transplantation, and such components are inmany cases harmful to the organ.

Although improvements to solutions have been made over the last severalyears, particularly as disclosed in U.S. Pat. Nos. 6,100,082, 6,046,046,and PCT application PCT/US98/19912, further improvements are stillneeded.

SUMMARY

The invention addresses deficiencies in the art by providing, in variousembodiments, improved systems, methods, compositions and solutions forperfusing an organ prior to transplantation. According to one aspect,the solutions include components adapted to preserving and maintaining aharvested organ ex-vivo in its functioning state under physiologic ornear physiologic conditions prior to its transplantation, whileminimizing reperfusion injury and ischemia to the organ while awaitingtransplantation.

According to another feature, the solutions include energy sources forthe organ while it is being prepared for and undergoing transplantation.Energy sources include, for example, one or more of carbohydratesources, components for synthesis of energy-rich molecules, such asphosphate sources, and other components. According to one embodiment,the solutions provide for amino acids for assisting the organ incellular protein synthesis during perfusion. According to oneembodiment, the solutions also provide components that help maintain theorgan's normal functionality during perfusion, such as cardiostimulants, insulin and other hormones, electrolytes, etc. The solutionsmay also be adapted to include drugs or other therapeutics forpreservation of the organ and/or patient care.

In another aspect, the invention provides for systems and methods forimproved delivery of preservation. In certain embodiments the solutionsare combined with blood products for perfusion. The solutions, systemsand methods are also adaptable to drug delivery systems. In certainembodiments, the solutions are combined with additional additives at thepoint of use.

In one embodiment, a composition for use in a solution for perfusing anorgan, such as a heart, is provided comprising one or morecarbohydrates, one or more organ stimulants, and a plurality of aminoacids that do not include asparagine, glutamine, or cysteine.

In another embodiment, a system for perfusing an organ, such as a heart,is provided comprising an organ and a substantially cell-freecomposition, where the compositions comprises one or more carbohydrates,one or more organ stimulants, and a plurality of amino acids that do notinclude asparagine, glutamine, or cysteine.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing and other objects and advantages of the invention will beappreciated more fully from the following further description thereof,with reference to the accompanying figures wherein:

FIG. 1 depicts an embodiment of a perfusion system using two solutionsfor maintaining a heart.

FIG. 2 depicts an embodiment of a perfusion system using two solutionsand also a priming solution.

FIG. 3 depicts an embodiment of a perfusion system using two solutions,a priming solution, and a composition or solution having sensitivematerial added prior to perfusion.

FIG. 4 depicts a chart demonstrating electrolyte stability for an organunder going perfusion according to an embodiment of the invention.

FIG. 5 depicts a chart demonstrating electrolyte stability for an organunder going perfusion according to another an embodiment of theinvention.

FIG. 6 depicts a chart demonstrating the arterial blood gas profile foran organ under going perfusion according to an embodiment of theinvention.

DETAILED DESCRIPTION

The invention addresses deficiencies in the prior art by providing invarious illustrated embodiments systems, methods, compositions, andsolutions for maintaining an organ in a functioning state ex vivo underphysiological or near physiological conditions. In one embodiment, theorgan is a heart and is maintained in a beating state at a physiologicor near physiologic temperature during perfusion of a perfusion fluid,which may include one or more of the solutions described herein.According to certain embodiments, solutions with particular solutes andconcentrations are selected and proportioned to enable the organ tofunction at physiologic or near physiologic conditions. For example,such conditions include maintaining organ function at or near aphysiological temperature and/or preserving an organ in a state thatpermits normal cellular metabolism, such as protein synthesis.

In certain embodiments solutions are formed from compositions bycombining components with a fluid, from more concentrated solutions bydilution, or from more dilute solutions by concentration. In exemplaryembodiments, suitable solutions include one or more energy sources, oneor more stimulants to assist the organ in continuing its normalphysiologic function prior to and during transplantation, and one ormore amino acids selected and proportioned so that the organ continuesits cellular metabolism during perfusion. Cellular metabolism includes,for example conducting protein synthesis while functioning duringperfusion. Some illustrative solutions are aqueous based, while otherillustrative solutions are non-aqueous, for example organicsolvent-based, ionic-liquid-based, or fatty-acid-based.

The solutions may include one or more energy-rich components to assistthe organ in conducting its normal physiologic function. Thesecomponents may include energy rich materials that are metabolizable,and/or components of such materials that an organ can use to synthesizeenergy sources during perfusion. Exemplary sources of energy-richmolecules include, for example, one or more carbohydrates. Examples ofcarbohydrates include monosaccharides, disaccharides, oligosaccharides,polysaccharides, or combinations thereof, or precursors or metabolitesthereof. While not meant to be limiting, examples of monosaccharidessuitable for the solutions include octoses; heptoses; hexoses, such asfructose, allose, altrose, glucose, mannose, gulose, idose, galactose,and talose; pentoses such as ribose, arabinose, xylose, and lyxose;tetroses such as erythrose and threose; and trioses such asglyceraldehyde. While not meant to be limiting, examples ofdisaccharides suitable for the solutions include (+)-maltose(4-O-(α-D-glucopyranosyl)-α-D-glucopyranose), (+)-cellobiose(4-O-(β-D-glucopyranosyl)-D-glucopyranose), (+)-lactose(4-O-(β-D-galactopyranosyl-β-D-glucopyranose), sucrose(2-O-(α-D-glucopyranosyl-β-D-fructofuranoside). While not meant to belimiting, examples of polysaccharides suitable for the solutions includecellulose, starch, amylose, amylopectin, sulfomucopolysaccharides (suchas dermatane sulfate, chondroitin sulfate, sulodexide, mesoglycans,heparan sulfates, idosanes, heparins and heparinoids), and glycogen. Insome embodiments, monossacharides, disaccharides, and polysaccharides ofboth aldoses, ketoses, or a combination thereof are used. One or moreisomers, including enantiomers, diastereomers, and/or tautomers ofmonossacharides, disaccharides, and/or polysaccharides, including thosedescribed and not described herein, may be employed in the solutionsdescribed herein. In some embodiments, one or more monossacharides,disaccharides, and/or polysaccharides may have been chemically modified,for example, by derivatization and/or protection (with protectinggroups) of one or more functional groups. In certain embodiments,carbohydrates, such as dextrose or other forms of glucose are preferred.

Other possible energy sources include adenosine triphosphate (ATP),co-enzyme A, pyruvate, flavin adenine dinucleotide (FAD), thiaminepyrophosphate chloride (co-carboxylase), β-nicotinamide adeninedinucleotide (NAD), β-nicotinamide adenine dinucleotide phosphate(NADPH), and phosphate derivatives of nucleosides, i.e. nucleotides,including mono-, di-, and tri-phosphates (e.g., UTP, GTP, GDF, and UDP),coenzymes, or other bio-molecules having similar cellular metabolicfunctions, and/or metabolites or precursors thereof. For example,phosphate derivatives of adenosine, guanosine, thymidine (5-Me-uridine),cytidine, and uridine, as well as other naturally and chemicallymodified nucleosides are contemplated.

In certain embodiments, one or more carbohydrates is provided along witha phosphate source, such as a nucleotide. The carbohydrate helps enablethe organ to produce ATP or other energy sources during perfusion. Thephosphate source may be provided directly through ATP, ADP, AMP, orother sources. In other illustrative embodiments, a phosphate isprovided through a phosphate salt, such as glycerophosphate, sodiumphosphate or other phosphate ions. A phosphate may include any formthereof in any ionic state, including protonated forms and forms withone or more counter ions.

The solutions may include one or more organ stimulants for assisting theorgan's normal physiologic function during perfusion. In someillustrative embodiments, where the transplanted organ is a heart,cardio stimulants are provided to enable the heart to continuefunctioning (e.g., continue beating) during perfusion andtransplantation. Such stimulants may include, for example,catecholamines, such as epinephrine and/or norepinephrine, whichfacilitate beating of the heart. Other cardio stimulants may be used,such as certain forms of peptides and/or polypeptides (e.g.,vasopressin, Anthropleurin-A and Anthropleurin-B), and/orβ1/β2-adrenoreceptor blocking agents (such as CGP 12177), buplinarol,pindolol, alprenolol, and cardiac glycosides. One or more naturalproducts may also be used, such as digitalis (digoxin), palustrin,and/or ferulic acid. Stimulants such as those mentioned above can beincluded with the solutions or added at the point of use by the user.

In some instances, additional components are provided to assist theorgan in conducting its metabolism during perfusion. These componentsinclude, for example, forms or derivatives of adenine and/or adenosine,which may be used for ATP synthesis, for maintaining endothelialfunction, and/or for attenuating ischemia and/or reperfusion injury.According to certain implementations, a magnesium ion source is providedwith a phosphate, and in certain embodiments, with adenosine to furtherenhance ATP synthesis within the cells of the perfused organ.

As noted, the solution may include one or more amino acids, preferably aplurality of amino acids, to support protein synthesis by the organ'scells. Suitable amino acids include, for example, any of thenaturally-occurring amino acids. The amino acids may be, in variousenantiomeric or diastereomeric forms. For example, solutions may employeither D- or L-amino acids, or a combination thereof, i.e. solutionsenantioenriched in more of the D- or L-isomer or racemic solutions.Suitable amino acids may also be non-naturally occurring or modifiedamino acids, such as citrulline, ornithine, homocystein, homoserine,β-amino acids such as β-alanine, amino-caproic acid, or combinationsthereof.

Certain exemplary solutions include some but not all naturally-occurringamino acids. In some embodiments, solutions include essential aminoacids. For example, a solution may be prepared with one or more or allof the following amino-acids:

Glycine Alanine Arginine Aspartic Acid Glutamic Acid HistidineIsoleucine Leucine Methionine Phenylalanine Proline Serine ThereonineTryptophan Tyrosine Valine Lysine acetate

In certain embodiments, non-essential and/or semi-essential amino acidsare not included in the solutions. For example, in some embodiments,asparagine, glutamine, and/or cysteine are not included. In otherembodiments, the solution contains one or more non-essential and/orsemi-essential amino acids. Accordingly, in other embodiments,asparagine, glutamine, and/or cysteine are included.

The solutions may also contain electrolytes, particularly calcium ionsfor facilitating enzymatic reactions, cardiac contractility, and/orcoagulation within the organ. Other electrolytes may be used, such assodium, potassium, chloride, sulfate, magnesium and other inorganic andorganic charged species or combinations thereof. It should be noted thatany component provided hereunder may be provided, where valence andstability permit, in an ionic form, in a protonated or unprotonatedform, in salt or free base form, or as ionic or covalent substituents incombination with other components that hydrolyze and make the componentavailable in aqueous solutions, as suitable and appropriate.

In certain embodiments, the solutions contain buffering components. Forexample, suitable buffer systems include 2-morpholinoethanesulfonic acidmonohydrate (MES), cacodylic acid, H₂CO₃/NaHCO₃ (pK_(a1)), citric acid(pK_(a3)), bis(2-hydroxyethyl)-imino-tris-(hydroxymethyl)-methane(Bis-Tris), N-carbamoylmethylimidino acetic acid (ADA),3-bis[tris(hydroxymethyl)methylamino]propane (Bis-Tris Propane)(pK_(a1)), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES),N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), imidazole,N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),3-(N-morpholino)propanesulphonic acid (MOPS), NaH₂PO₄/Na₂HPO₄ (pK_(a2)),N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES),N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (HEPES),N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid)(HEPPSO), triethanolamine, N-[tris(hydroxymethyl)methyl]glycine(Tricine), tris hydroxymethylaminoethane (Tris), glycineamide,N,N-bis(2-hydroxyethyl)glycine (Bicine), glycylglycine (pKa2_(a2)),N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), or acombination thereof. In some embodiments, the solutions contain sodiumbicarbonate, potassium phosphate, or TRIS buffer.

The solutions may include other components to help maintain the organand protect it against ischemia, reperfusion injury and other illeffects during perfusion. In certain exemplary embodiments thesecomponents may include hormones (e.g., insulin), vitamins (e.g., anadult multi-vitamin, such as multi-vitamin MVI-Adult™), and/or steroids(e.g., dexamethasone and SoluMedrol™).

In another aspect, a blood product is provided with the solution tosupport the organ during metabolism. Exemplary suitable blood productsmay include whole blood, and/or one or more components thereof such asblood serum, plasma, albumin, and red blood cells. In embodiments wherewhole blood is used, the blood may be passed through a leukocyte andplatelet depleting filter to reduce pyrogens, antibodies and/or otheritems that may cause inflammation in the organ. Thus, in someembodiments, the solution employs whole blood that has been at leastpartially depleted of leukocytes and/or whole blood that has been atleast partially depleted of platelets.

The solutions are preferably provided at a physiological temperature andmaintained thereabout throughout perfusion and recirculation. As usedherein, “physiological temperature” is referred to as temperaturesbetween about 25° C. and about 37° C., for example, between about 30° C.and about 37° C., such as between about 34° C. and about 37° C.

Table 1 sets forth components that may be used in a preservativesolution for preserving an organ as described herein.

TABLE 1 Component of Exemplary Composition for a Preservative SolutionExemplary Concentration Component Ranges in a preservative solutionAlanine about 1 mg/L-about 10 g/L Arginine about 1 mg/L-about 10 g/LAsparagine about 1 mg/L-about 10 g/L Aspartic Acid about 1 mg/L-about 10g/L Cysteine about 1 mg/L-about 10 g/L Cystine about 1 mg/L-about 10 g/LGlutamic Acid about 1 mg/L-about 10 g/L Glutamine about 1 mg/L-about 10g/L Glycine about 1 mg/L-about 10 g/L Histidine about 1 mg/L-about 10g/L Hydroxyproline about 1 mg/L-about 10 g/L Isoleucine about 1mg/L-about 10 g/L Leucine about 1 mg/L-about 10 g/L Lysine about 1mg/L-about 10 g/L Methionine about 1 mg/L-about 10 g/L Phenylalanineabout 1 mg/L-about 10 g/L Proline about 1 mg/L-about 10 g/L Serine about1 mg/L-about 10 g/L Threonine about 1 mg/L-about 10 g/L Tryptophan about1 mg/L-about 10 g/L Tyrosine about 1 mg/L-about 10 g/L Valine about 1mg/L-about 10 g/L Adenine about 1 mg/L-about 10 g/L ATP about 10ug/L-about 100 g/L Adenylic Acid about 10 ug/L-about 100 g/L ADP about10 ug/L-about 100 g/L AMP about 10 ug/L-about 100 g/L Ascorbic Acidabout 1 ug/L-about 10 g/L D-Biotin about 1 ug/L-about 10 g/L VitaminD-12 about 1 ug/L-about 10 g/L Cholesterol about 1 ug/L-about 10 g/LDextrose (Glucose) about 1 g/L-about 150 g/L Multi-vitamin Adult about 1mg/L-about 20 mg/L or 1 unit vial Epinephrine about 1 ug/L-about 1 g/LFolic Acid about 1 ug/L-about 10 g/L Glutathione about 1 ug/L-about 10g/L Guanine about 1 ug/L-about 10 g/L Inositol about 1 g/L-about 100 g/LRiboflavin about 1 ug/L-about 10 g/L Ribose about 1 ug/L-about 10 g/LThiamine about 1 mg/L-about 10 g/L Uracil about 1 mg/L-about 10 g/LCalcium Chloride about 1 mg/L-about 100 g/L NaHCO₃ about 1 mg/L-about100 g/L Magnesium sulfate about 1 mg/L-about 100 g/L Potassium chlorideabout 1 mg/L-about 100 g/L Sodium glycerophosphate about 1 mg/L-about100 g/L Sodium Chloride about 1 mg/L-about 100 g/L Sodium Phosphateabout 1 mg/L-about 100 g/L Insulin about 1 IU-about 150 IU Serum albuminabout 1 g/L-about 100 g/L Pyruvate about 1 mg/L-about 100 g/L Coenzyme Aabout 1 ug/L-about 10 g/L Serum about 1 ml/L-about 100 ml/L Heparinabout 500 U/L-about 1500 U/L SoluMedrol ™ about 200 mg/L-about 500 mg/LDexamethasone about 1 mg/L-about 1 g/L FAD about 1 ug/L-about 10 g/LNADP about 1 ug/L-about 10 g/L adenosine about 1 mg/L-about 10 g/Lguanosine about 1 mg/L-about 10 g/L GTP about 10 ug/L-about 100 g/L GDPabout 10 ug/L-about 100 g/L GMP about 10 ug/L-about 100 g/L

The solutions described herein may be prepared from compositions havingone or more components such as those described above in concentrationssuch as those described above. An exemplary embodiment of a compositionadaptable for use in organ perfusion includes one or more componentsselected from Table 2. The amounts provided describe preferred amountsrelative to other components in the table and may be scaled to providecompositions of sufficient quantity. In some embodiments, the amountslisted in Table 2 can vary by ±about 10% and still be used in thesolutions described herein.

TABLE 2 Component of Exemplary Composition for a Preservative SolutionComponent Amount Adenosine about 675 mg-about 825 mg Calcium Chloridedihydrate about 2100 mg-about 2600 mg Glycine about 315 mg-about 385 mgL-Alanine about 150 mg-about 200 mg L-Arginine about 600 mg-about 800 mgL-Aspartic Acid about 220 mg-about 270 mg L-Glutamic Acid about 230 mgabout 290 mg L-Histidine about 200 mg-about 250 mg L-Isoleucine about100 mg about 130 mg L-Leucine about 300 mg-about 380 mg L-Methionineabout 50 mg-about 65 mg L-Phenylalanine about 45 mg-about 60 mgL-Proline about 110 mg-about 140 mg L-Serine about 80 mg-about 105 mgL-Thereonine about 60 mg-about 80 mg L-Tryptophan about 30 mg-about 40mg L-Tyrosine about 80 mg-about 110 mg L-Valine about 150 mg-about 190mg Lysine Acetate about 200 mg-about 250 mg Magnesium SulfateHeptahydrate about 350 mg-about 450 mg Potassium Chloride about 15mg-about 25 mg Sodium Chloride about 1500 mg-about 2000 mg Dextroseabout 25 g-about 120 g Epinephrine about 0.25 mg-about 1.0 mg Insulinabout 75 Units-about 150 Units MVI-Adult ™ 1 unit vial SoluMedrol ™about 200 mg-about 500 mg Sodium Bicarbonate about 10-about 25 mEq

A solution may be prepared combining components selected by a user withan aqueous fluid to form a preservative solution. In an illustrativeembodiment, one or more components from Table 2 are combined in therelative amounts listed therein per about 1000 mL of aqueous fluid toform a preservative solution for perfusing an organ, such as a heart. Insome embodiments the quantity of aqueous fluid can vary ±about 10%.

In another aspect, solutions may be administered in combinations asdesired by a medical operator. FIG. 1 is a conceptual diagram of asystem 10 for perfusing a heart 12 according to an illustrativeembodiment of the invention. As shown, the illustrative system 10includes two solutions, a first solution 13 and a second solution 15,which are joined through fluid line 14 and become part of a perfusioncircuit that includes lines 16, 18 and 20. The fluid lines interconnecta perfusion operating system 22 and a mixing chamber 24, with solutions13 and 15 to perfuse the heart 12. In some embodiments, the system 10 issterilized prior to use. In operation, the solutions 13 and 15 feed tothe mixing chamber 24 via the conduit 14. Solutions 13 and 15 mix with aperfusion fluid that circulates in the perfusion circuit. The perfusionfluid may be blood or synthetic blood based. Perfusion fluidrecirculates from the heart 12 via the fluid conduit 18, the perfusionoperating system 22, and the fluid conduit 20 to the mixing chamber 24.There the recirculated fluid is mixed with solutions 13 and 15 and fedto the heart via the conduit 16. Since the heart 12 is maintained in abeating state, it continues to received fluid from the conduit 16 andfeed it back via the conduit 18 for remixing in the mixing chamber 24.

FIG. 2 is another conceptual drawing depicting a further illustrativeembodiment of the perfusion system 10. As shown, the illustrativeembodiment of FIG. 2 includes a priming solution 17 applied to thesystem prior to addition of a perfusion fluid. As shown in FIG. 2, thepriming solution 17 flows into line 26 to provide the priming solution17 to the system prior to perfusing the heart 12 with a perfusion fluidcontaining solutions 13 and 15. The priming solution 17 may include, forexample, any or all of the components included in the solutions 13 and15.

In other illustrative embodiments, the solutions 15 and/or 17 may beprovided systematically to optimize the effectiveness of the system. Forexample, as shown in FIG. 3, the solution 13 may include a compositionor solution 19, which may include components that are sensitive tosterilization and other techniques used to prepare the solution 13 forperfusion. For example, the composition or solution 19 may includeinsulin or other bio-components, organic compounds, and/or biologicalmolecules that would decompose or degrade when sterilized, such as viaan autoclave. In this instance, as shown in FIG. 3, the composition orsolution 19 may be added to the solution 13 prior to or during perfusionof the donor organ. As shown in FIG. 3, the composition or solution 19may be applied to the solution 13 through a fluid conduit 28. Thecomposition or solution 19 may also be applied to fluid conduit 14(though not shown in FIG. 3), and then mixed with the perfusion fluid inchamber 24 just prior to application to the conduit 16.

In some illustrative embodiments, the solutions described herein arecombined with a blood product, such as whole blood or componentsthereof, which may also be oxygenated, to give a perfusion fluid.Additionally, synthetic blood products may be used as a substitute or incombination with blood products. Such combined solutions may be includedin a perfusion circuit, such as those shown in FIGS. 1 to 3.

In other illustrative embodiments, one of the composition or solutions13, 15, 17, or 19 may include one or more organ stimulants, such ascardio stimulants, and/or other components. The other components mayinclude, for example, adenosine, a magnesium ion source, one or morephosphates, calcium ions, etc. In embodiments utilizing two or moresolutions, such as those in FIGS. 1 to 3, the solution 15 may includeone or more carbohydrates and may also include a phosphate source.According to some illustrative embodiments, the perfusion fluid includesmore than one composition or solution 13, 15, 17 or 19 to avoidprecipitation of calcium phosphate in embodiments where calcium ions andphosphate ions are used.

In one embodiment, a maintenance solution is made from the combinationof a first solution, substantially formed from one or more amino acids,and a second solution, substantially formed from one or morecarbohydrates, such as dextrose or glucose. The maintenance solution mayalso have additives, such as those described herein, administered at thepoint of use just prior to infusion into the organ perfusion system. Forexample, additional additives that can be included with the solution oradded at the point of use by the user include hormones and steroids,such as dexamethasone and insulin, as well as vitamins, such as an adultmulti-vitamin, for example adult multivitamins for infusion, such asMVI-Adult™. Additional small molecules and large bio-molecules may alsobe included with the solution or added at the point of use by the user,for example, therapeutics and/or components typically associated withblood or blood plasma, such as albumin.

In some embodiments, therapeutics that may be included in thecompositions, solutions, and systems described herein include hormones,such as thyroid hormones, for example T₃ and/or T₄ thyroid hormones.Further therapeutics that may be included include drugs such asanti-arrhythmic drugs, for example, for heart therapy, and betablockers. For instance, in certain embodiments, one or more thyroidhormones, one or more anti-arrhythmic drugs, and one or more betablockers are added to the first solution, the second solution, and/orthe maintenance solution either before or during perfusion of the organ.The above therapeutics may also be added directly to the system, forexample to the perfusion circuit before or during perfusion of theorgan.

Table 3 sets forth an exemplary first solution, comprising a tissueculture media having the components identified in Table 3 and combinedwith an aqueous fluid, which may be used as to perfuse an organ asdescribed herein. The amounts of components listed in Table 3 arerelative to each other and to the quantity of aqueous solution used. Insome embodiments, about 500 mL of aqueous fluid is used. In otherembodiments about 1 L of aqueous fluid is used. For example, combinationof about 500 mL of first solution with 500 mL of second solution affordsa maintenance solution of about 1 L. In some embodiments, the quantityof aqueous solution can vary ±about 10%. The component amounts and thequantity of aqueous solution may be scaled as appropriate for use. ThepH of the first solution, in this embodiment, may be adjusted to beabout 7.0 to about 8.0, for example about 7.3 to about 7.6.

TABLE 3 Composition of Exemplary First Solution (about 500 mL aqueoussolution) Tissue Culture Component Amount Specification Adenosine 750 mg±about 10% Calcium Chloride dihydrate 2400 mg  ±about 10% Glycine 350 mg±about 10% L-Alanine 174 mg ±about 10% L-Arginine 700 mg ±about 10%L-Aspartic Acid 245 mg ±about 10% L-Glutamic Acid 258 mg ±about 10%L-Histidine 225 mg ±about 10% L-Isoleucine 115.5 mg  ±about 10%L-Leucine 343 mg ±about 10% L-Methionine  59 mg ±about 10%L-Phenylalanine  52 mg ±about 10% L-Proline 126 mg ±about 10% L-Serine 93 mg ±about 10% L-Thereonine  70 mg ±about 10% L-Tryptophan  35 mg±about 10% L-Tyrosine  92 mg ±about 10% L-Valine 171.5 mg  ±about 10%Lysine Acetate 225 mg ±about 10% Magnesium Sulfate Heptahydrate 400 mg±about 10% Potassium Chloride  20 mg ±about 10% Sodium Chloride 1750 mg ±about 10%

Since amino acids are the building blocks of proteins, the uniquecharacteristics of each amino acid impart certain important propertieson a protein such as the ability to provide structure and to catalyzebiochemical reactions. The selection and concentrations of the aminoacids provided in the first solution provide support of normalphysiologic functions such as metabolism of sugars to provide energy,regulation of protein metabolism, transport of minerals, synthesis ofnucleic acids (DNA and RNA), regulation of blood sugar and support ofelectrical activity, in addition to providing protein structure.Additionally, the concentrations of specific amino acids found in thefirst solution can be used to predictably stabilize the pH of the firstsolution and/or other solutions and fluids to which the first solutionmay be added, for example the maintenance solution and/or the perfusionfluid.

Table 4 provides an exemplary second solution 15, comprising a solutionwhich may be used with the systems of FIGS. 1-3. The second solution 15includes dextrose and an aqueous solvent. In some embodiments, thesecond solution 15 further includes sodium glycerol phosphate. Theamount of components in Table 5 is relative to the amount of aqueoussolvent (about 500 mL) and may be scaled as appropriate. In someembodiments, the quantity of aqueous solvent varies ±about 10%.

TABLE 4 Components of Exemplary Second Solution (about 500 mL) ComponentAmount Specification Dextrose 40 g ±about 10%

Additional components may be included in the first or second solution,the maintenance solution, and/or the priming solution and may includeone or more of those set forth in Table 5. These additional orsupplemental components may be added individually, in variouscombinations, or all at once as a composition. For example, in certainembodiments, the epinephrine, insulin, and MVI-Adult™, listed in Table5, are added to the maintenance solution. In another example, theSoluMedrol™ and the sodium bicarbonate, listed in Table 5, are added tothe priming solution. The additional components may also be combined inone or more combinations or all together and placed in solution beforebeing added to the first or second solution or to the maintenancesolution. These additional components may be degraded or otherwiseinactivated if subjected to sterilization, and, as such, may beappropriately applied directly to the perfusion circuit or to a first 13or second 15 solution after sterilization, such as described with regardto the composition or solution 19 of FIG. 3. In some embodiments,dexamethasone may also be added to solutions described herein. Thecomponent amounts listed in Table 5 are relative to each other and tothe amounts of components in Table 3, Table 4 and/or Table 6 and/or tothe amount of aqueous solution used in the first or second solution, themaintenance solution, or the priming solution and may be scaled asappropriate for the amount required.

TABLE 5 Exemplary Supplemental Components Added Prior to Use ComponentAmount Type Function Specification Epinephrine about 0.50 mgCatecholamine Maintains Vascular ±about 10% Hormone Tone, BasalCatecholamine Levels for Cardiac Function Insulin about 100 UnitsHormone Facilitates Glucose ±about 10% Absorption MVI-Adult ™ 1 unitvial Vitamin Source of antioxidants ±about 10% SoluMedrol ™ about 250 mgin Steroid ±about 10% about 4 mL water Sodium about 20 mEq BufferMaintains pH ±about 10% Bicarbonate

As described above, in certain illustrative embodiments, solutions areused to perfuse an organ prior to transplantation. In certainembodiments one or more solutions are applied to maintain the organ, anda separate solution is applied to prime the organ prior to applying oneor more maintenance solutions. Certain exemplary components for primingsolutions are identified in Table 6. In some embodiments, the primingsolutions includes one or more carbohydrates. The component amounts inTable 6 are relative to each other and to the amount of aqueous solvent(about 500 mL) and may be scaled as appropriate. In certain embodiments,the quantity of aqueous solvent varies ±about 10%.

TABLE 6 Composition of Exemplary Priming Solution (about 500 mL aqueoussolution) Component Amount Specification Mannitol 12.5 g ±about 10%Sodium Chloride 4.8 g ±about 10% Potassium Chloride 185 mg ±about 10%Magnesium Sulfate heptahydrate 185 mg ±about 10% Sodium Glycerophosphate900 mg ±about 10%

In some illustrative embodiments, the invention combines solutions asdescribed herein in desired sequences to provide for improvedphysiological performance of the organ. Although the first and secondsolutions are shown above, and referenced as solutions 13 and 15 inFIGS. 1-3, the solutions may be divided into any number of solutions,their positions interchanged in system 10, and applied in any mannernecessary to maintain the functioning organ.

In some illustrative embodiments, the solutions contemplated herein maybe adapted for use with an organ perfusion system such as that describedin U.S. Pat. Nos. 6,100,082 or 6,046,046, or PCT applicationPCT/US98/19912, the specifications of which are hereby incorporated byreference in their entirety.

Certain embodiments of the first solution include epinephrine and aplurality of amino acids. In certain embodiments, the first solutionincludes electrolytes, such as calcium and magnesium. The first solutionmay also include other components such as adenosine and/or one or morecomponents from Table 3. In certain embodiments a second solutionincludes one or more carbohydrates and may also include a phosphatesource. The second solution is typically maintained at a pH of about 5.0to about 6.5, for example of about 5.5 to about 6.0.

In certain embodiments methods for perfusing an organ are providedthrough the use of solutions described herein, such as through thesystems illustrated in FIGS. 1 to 3. As shown in the figures, perfusionmay occur through a beating organ and may take place at physiologicaltemperature. In certain embodiments the methods include providing afluid for perfusing the organ, the fluid including a phosphate source,one or more organ stimulants, such as epinephrine, and one or morecarbohydrates. In certain embodiments the fluid may be perfused throughthe organ and the organ is maintained in a beating state without the useof an applied vasopressor to effect the circulation of the fluid. Insome embodiments, the organ is a heart.

In one illustrative embodiment, the system described in FIGS. 1-3 isused to perfuse an organ. In this embodiment, the system has a port fora priming solution that includes the priming solution shown in Table 6,a first chamber that contains a first solution having the componentsdescribed above in Table 3, a port for a supplemental composition orsolution having the components described in Table 5, and a secondchamber containing a second solution having the components shown inTable 4. The priming solution may also be contained in a third chamber.The first solution, second solution, and the priming solution aresterilized through an autoclave, for example, to a sterility assurancelevel of about 10⁻⁶, then the supplemental composition or solution isadded to the first solution. The perfusion system is then primed withthe priming solution for about 10 minutes. In some embodiments, thepriming is through perfusion in the direction of normal flow of fluidthrough the organ. In other embodiments, the priming is throughretrograde perfusion, i.e., having a flow direction that is counterdirectional to the normal flow of fluid through the organ. Afterpriming, the remaining solutions are combined and infused into theperfusion system at a flow-rate selected to replace necessary substratesas they are consumed by the organ. In some embodiments, the flow rateapproximates physiologic fluid flow through the organ. Fluid flow ratesduring heart priming are about 1 L/min, while solution infusion ratesduring heart perfusion are about 10 mL/hr to about 40 mL/hr. Optionally,an additional solution containing a blood product is provided throughwhich the blood product is combined with the circulating fluid and usedto perfuse the organ.

In one embodiment, a composition for use in a solution for perfusing anorgan, such as a heart, is provided comprising one or morecarbohydrates, one or more organ stimulants, and a plurality of aminoacids that do not include asparagine, glutamine, or cysteine. Thecomposition may also include other substances, such as those used insolutions described herein.

In another embodiment, a system for perfusing an organ, such as a heart,is provided comprising an organ and a substantially cell-freecomposition, comprising one or more carbohydrates, one or more organstimulants, and a plurality of amino acids that do not includeasparagine, glutamine, or cysteine. Substantially cell-free includessystems that are substantially free from cellular matter; in particular,systems that are not derived from cells. For example, substantiallycell-free includes compositions and solutions prepared from non-cellularsources.

In another aspect, the solutions and systems may be provided in the formof a kit that includes one or more organ maintenance solutions. Anexemplary maintenance solution may include components identified abovein one or more fluid solutions for organ perfusion. In certainembodiments, the maintenance solution may include multiple solutions,such as a first solution and a second solution and/or a supplementalcomposition or solution, or may include dry components that may beregenerated in a fluid to form one or more solutions for organperfusion. The kit may also comprise one or more concentrated solutionswhich, on dilution, provide a first, second, and/or supplementalsolution as described herein. The kit may also include a primingsolution. In an exemplary embodiment, the maintenance solution includesa first solution and second solution such as those described above, anda priming solution such as that described above.

In certain embodiments, the kit is provided in a single package, whereinthe kit includes one or more solutions (or components necessary toformulate the one or more solutions by mixing with an appropriatefluid), and instructions for sterilization, flow and temperature controlduring perfusion and use and other information necessary or appropriateto apply the kit to organ perfusion. In certain embodiments, a kit isprovided with only a single solution (or set of dry components for usein a solution upon mixing with an appropriate fluid), and the singlesolution (or set of dry components) is provided along with a set ofinstructions and other information or materials necessary or useful tooperate the solution in a perfusion apparatus or system.

In another aspect, the systems, solutions and methods may be used todeliver therapeutics to an organ during perfusion. For example, one ormore of the solutions and/or systems described above may include one ormore drugs, biologics, gene therapy vectors, or other therapeutics whichare delivered to the organ during perfusion. Suitable exemplarytherapeutics may include drugs, biologics, or both. Suitable drugs mayinclude, for example, anti fungals, anti-microbials or anti-biotics,anti-inflammatories, anti-proliferatives, anti-virals, steroids,retinoids, NSAIDs, vitamin D3 and vitamin D3 analogs, calcium channelblockers, complement neutralizers, ACE inhibitors, immuno-suppressants,and other drugs. Suitable biologics may include proteins; suitablebiologics may also include vectors loaded with one or more genes forgene therapy application.

For example, suitable steroids include but are not limited to androgenicand estrogenic steroid hormones, androgen receptor antagonists and5-α-reductase inhibitors, and corticosteroids. Specific examples includebut are not limited to alclometasone, clobetasol, fluocinolone,fluocortolone, diflucortolone, fluticasone, halcinonide, mometasone,prednisone, prednisolone, methylprednisolone, triamcinolone,betamethasone, and dexamethasone, and various esters and acetonidesthereof.

Suitable retinoids include but are not limited to retinol, retinal,isotretinoin, acitretin, adapalene, tazarotene, and bexarotene.

Suitable NSAIDs include but are not limited to naproxen, suprofen,ketoprofen, ibuprofen, flurbiprofen, diclofenac, indomethacin,celecoxib, and rofecoxib.

Suitable vitamin D3 analogues include but are not limited todoxercalciferol, seocalcitol, calcipotriene, tacalcitol, calcitriol,ergocalciferol, and calcifediol.

Suitable anti-viral agents include but are not limited to trifluridine,cidofovir, acyclovir, penciclovir, famciclovir, valcyclovir,gancyclovir, and docosanol.

Suitable human carbonic anhydrase inhibitors include but are not limitedto methazoliamide, acetazolamide, and dorzolamide.

Suitable anti-proliferative agents include but are not limited to 5-FU,taxol, daunorubicin, and mitomycin.

Suitable antibiotic (antimicrobial) agents include but are not limitedto bacitracin, chlorhexidine, chlorhexidine digluconate, ciprofloxacin,clindamycin, erythromycin, gentamicin, lomefloxacin, metronidazole,minocycline, moxifloxacin, mupirocin, neomycin, ofloxacin, polymyxin B,rifampicin, ruflozacin, tetracycline, tobramycin, triclosan, andvancomycin. The antiviral and antibacterial prodrugs described hereinmay be used to treat appropriately responsive systemic infections.

Certain experimental data are available to describe certain embodimentsof the solutions described herein and their use in organ perfusion.Certain data are set for in FIGS. 4-6. FIG. 4 depicts a chartdemonstrating electrolyte stability for an organ under going perfusionaccording to an embodiment of the invention. In the embodimentassociated with FIG. 4, the organ is a heart wherein perfusion isconducted analogous to physiological conditions; i.e. a solution asdescribed herein is perfused in the typical physiological direction outof the aorta. The rate of perfusion is approximately 30 mL/hr. As can beseen from FIG. 4, the levels of various electrolytes: sodium, potassium,calcium, and chloride ions, as well as dissolved glucose, remain atstable levels throughout the course of perfusion, from before the organis connected to the perfusion system (the pre-instrumentation (PI)period) to six hours after connection of the organ.

FIG. 5 depicts a chart demonstrating electrolyte stability for an organunder going perfusion according to another embodiment of the invention.In the embodiment associated with FIG. 5, the organ is a heart whereinperfusion is conducted, in one respect, in an atypical fashion fromphysiological conditions; that is, a solution as described herein isperfused in the reverse physiological direction into the aorta. Such aretrograde perfusion perfuses the coronary sinus. In some embodiments,the left side of the heart remains empty. The rate of perfusion isapproximately 30 mL/hr. As can be seen from FIG. 5, the levels ofvarious electrolytes: sodium, potassium, calcium, and chloride ions, aswell as dissolved glucose, remain at stable levels throughout the courseof perfusion, from before the organ is connected to the perfusion system(the pre-instrumentation (PI) period) to six hours after connection ofthe organ. FIG. 5 also demonstrates that the levels of the electrolytesand glucose remain at levels similar to those for the base line (BL)normal physiological state for the organ.

FIG. 6 depicts a chart demonstrating the arterial blood gas profile foran organ under going perfusion according to another embodiment of theinvention. As can be seen from FIG. 6, the levels of various bloodgasses: carbon dioxide and oxygen, and pH remain at stable levelsthroughout the six hour course of perfusion. FIG. 6 also demonstratesthat the levels of carbon dioxide, oxygen, and pH remain at levelssimilar to those for two base line (BL) measurements for the normalphysiological state for the organ. FIGS. 4-6 demonstrate the ability ofthe present systems and methods to maintain an organ under stablephysiological or near physiological conditions.

This application incorporates by reference the specification from eachof the following applications: U.S. application Ser. No. 09/534,092,filed on Mar. 23, 2000, PCT/US98/19912, filed on Sep. 23, 1998, U.S.application Ser. No. 09/054,698 filed on Apr. 3, 1998, and U.S.application Ser. No. 08/936,062 filed on Sep. 23, 1997.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the forgoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Forexample, a variety of systems and/or methods may be implemented based onthe disclosure and still fall within the scope of the invention. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A perfusion system configured for maintaining anex-vivo heart in a functioning state under physiological or nearphysiological conditions comprising: a first chamber containing a firstsolution comprising one or more cardio stimulants selected from thegroup consisting of catecholamines, peptides, polypeptides,β1/β2-adrenoreceptor blocking agents, buplinarol, pindolol, alprenolol,cardiac glycosides, digitalis, palustrin, and ferulic acid; and aplurality of amino acids selected from the group consisting of alanine,arginine, aspartic acid, glutamic acid, histidine, isoleucine, leucine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine, and lysine that do not include asparagine, glutamine,and cysteine; a second chamber containing a second solution comprisingone or more carbohydrates selected from the group consisting ofmonosaccharides, disaccharides, oligosaccharides, and polysaccharides;and a third chamber containing a priming solution comprising mannitol,sodium chloride, potassium chloride, magnesium sulfate heptahydrate, andsodium glycerophosphate.
 2. The perfusion system of claim 1, wherein atleast one of the first solution, second solution, or priming solution issterilized prior to using the solutions to perfuse the heart.
 3. Theperfusion system of claim 1, wherein the one or more cardio stimulantsincludes epinephrine.
 4. The perfusion system of claim 1, wherein theone or more carbohydrates in the second solution includes dextrose. 5.The perfusion system of claim 1, wherein the first solution includesadenosine.
 6. The perfusion system of claim 1, wherein the firstsolution includes insulin.
 7. The perfusion system of claim 1, whereinthe first solution includes an immuno-suppressant.
 8. The perfusionsystem of claim 1, wherein the first solution includes a multi-vitamincomposition.
 9. The perfusion system of claim 1, further comprising oneor more electrolytes in the first solution.
 10. The perfusion system ofclaim 9, wherein the one or more electrolytes includes calcium.
 11. Theperfusion system of claim 1, wherein the solutions are combined with ablood product prior to perfusing the heart.
 12. The perfusion system ofclaim 1, further comprising a therapeutic agent.
 13. A kit for use in aperfusion system that is configured for maintaining an ex-vivo heart ina functioning state under physiological or near physiologicalconditions, the kit comprising: a first solution comprising one or morecarbohydrates selected from the group consisting of monosaccharides,disaccharides, oligosaccharides, and polysaccharides, a second solutioncomprising one or more cardio stimulants selected from the groupconsisting of catecholamines, peptides, polypeptides,δ1/β2-adrenoreceptor blocking agents, buplinarol, pindolol, alprenolol,cardiac glycosides, digitalis, palustrin, and ferulic acid; and aplurality of amino acids selected from the group consisting of alanine,arginine, aspartic acid, glutamic acid, histidine, isoleucine, leucine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine, and lysine that do not include asparagine, glutamine,and cysteine, and a priming solution comprising mannitol, sodiumchloride, potassium chloride, magnesium sulfate heptahydrate, and sodiumglycerophosphate, and a package.